42 research outputs found

    Off-target effects of SGLT2 blockers: empagliflozin does not inhibit Na+/H+ exchanger-1 or lower [Na+]i in the heart

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    Aims: Empagliflozin (EMPA) is a potent inhibitor of the renal sodium-glucose cotransporter 2 (SGLT2) and an effective treatment for type-2 diabetes. In patients with diabetes and heart failure, EMPA has cardioprotective effects independent of improved glycaemic control, despite SGLT2 not being expressed in the heart. A number of non-canonical mechanisms have been proposed to explain these cardiac effects, most notably an inhibitory action on cardiac Na+/H+ exchanger 1 (NHE1), causing a reduction in intracellular [Na+] ([Na+]i). However, at resting intracellular pH (pHi), NHE1 activity is very low and its pharmacological inhibition is not expected to meaningfully alter steady-state [Na+]i. We re-evaluate this putative EMPA target by measuring cardiac NHE1 activity. Methods and results: The effect of EMPA on NHE1 activity was tested in isolated rat ventricular cardiomyocytes from measurements of pHi recovery following an ammonium pre-pulse manoeuvre, using cSNARF1 fluorescence imaging. Whereas 10 µM cariporide produced near-complete inhibition, there was no evidence for NHE1 inhibition with EMPA treatment (1, 3, 10 or 30 µM). Intracellular acidification by acetate-superfusion evoked NHE1 activity and raised [Na+]i, reported by sodium binding benzofuran isophthalate (SBFI) fluorescence, but EMPA did not ablate this rise. EMPA (10 µM) also had no significant effect on the rate of cytoplasmic [Na+]i-rise upon superfusion of Na+-depleted cells with Na+-containing buffers. In Langendorff-perfused mouse, rat and guinea pig hearts, EMPA did not affect [Na+]i at baseline nor pHi recovery following acute acidosis, as measured by 23Na triple quantum filtered NMR and 31P NMR, respectively. Conclusions Our findings indicate that cardiac NHE1 activity is not inhibited by EMPA (or other SGLT2i’s) and EMPA has no effect on [Na+]i over a wide range of concentrations, including the therapeutic dose. Thus, the beneficial effects of SGLT2i’s in failing hearts should not be interpreted in terms of actions on myocardial NHE1 or intracellular [Na+]. Translational Perspective: Heart failure remains a huge clinical burden. Clinical trials of SGLT2 inhibitors in patients with diabetes and heart failure have reported highly significant cardiovascular benefit that appears independent of improved glycaemic control. As SGLT2 is not expressed in the heart, the mechanism by which SGLT2 inhibitors are cardioprotective remains unknown. Understanding this mechanism is clearly essential as the use of SGLT2 inhibitors in non-diabetics is increasing and a better understanding may allow refinement of therapeutic approaches in both HFpEF and HFrEF. One suggested mechanism that has received significant attention, inhibition of cardiac Na+/H+ exchanger, is investigated here

    A protocol for dual calcium-voltage optical mapping in murine sinoatrial preparation with optogenetic pacing

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    Among the animal models for studying the molecular basis of atrial and sinoatrial node (SAN) biology and disease, the mouse is a widely used species due to its feasibility for genetic modifications in genes encoding ion channels or calcium handling and signaling proteins in the heart. It is therefore highly valuable to develop robust methodologies for studying SAN and atrial electrophysiological function in this species. Here, we describe a protocol for performing dual calcium-voltage optical mapping on mouse sinoatrial preparation (SAP), in combination with an optogenetic approach, for studying SAP membrane potential, intracellular Ca2+ transients, and pacemaker activity. The protocol includes the details for preparing the intact SAP, robust tissue dual-dye loading, light-programmed pacing, and high-resolution optical mapping. Our protocol provides an example of use of the combination of optogenetic and optical mapping techniques for investigating SAP membrane potential and intracellular Ca2+ transients and pacemaker activity with high temporal and spatial resolution in specific cardiac tissues. Thus, our protocol provides a useful tool for studying SAP physiology and pathophysiology in mice

    A Regional Reduction in Ito and IKACh in the Murine Posterior Left Atrial Myocardium Is Associated with Action Potential Prolongation and Increased Ectopic Activity.

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    BACKGROUND: The left atrial posterior wall (LAPW) is potentially an important area for the development and maintenance of atrial fibrillation. We assessed whether there are regional electrical differences throughout the murine left atrial myocardium that could underlie regional differences in arrhythmia susceptibility. METHODS: We used high-resolution optical mapping and sharp microelectrode recordings to quantify regional differences in electrical activation and repolarisation within the intact, superfused murine left atrium and quantified regional ion channel mRNA expression by Taqman Low Density Array. We also performed selected cellular electrophysiology experiments to validate regional differences in ion channel function. RESULTS: Spontaneous ectopic activity was observed during sustained 1Hz pacing in 10/19 intact LA and this was abolished following resection of LAPW (0/19 resected LA, P<0.001). The source of the ectopic activity was the LAPW myocardium, distinct from the pulmonary vein sleeve and LAA, determined by optical mapping. Overall, LAPW action potentials (APs) were ca. 40% longer than the LAA and this region displayed more APD heterogeneity. mRNA expression of Kcna4, Kcnj3 and Kcnj5 was lower in the LAPW myocardium than in the LAA. Cardiomyocytes isolated from the LAPW had decreased Ito and a reduced IKACh current density at both positive and negative test potentials. CONCLUSIONS: The murine LAPW myocardium has a different electrical phenotype and ion channel mRNA expression profile compared with other regions of the LA, and this is associated with increased ectopic activity. If similar regional electrical differences are present in the human LA, then the LAPW may be a potential future target for treatment of atrial fibrillation
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